The overall goal of this project is to characterize the molecular mechanisms of HCN and hERG channel gating. HCN channels conduct the pacemaker current (If), and hERG channels conduct the rapid delayed rectifier current IKr. The voltage and time-dependent gating of these currents contribute to the rhythmic firing of cardiac pacemaker cells. HERG channels are opened by membrane depolarization, coupled to outward motion of the S4 voltage sensor. In contrast, HCN channels are opened by membrane hyperpolarization, coupled to inward motion of the S4 domain. We hypothesize that the opposite polarity of coupling between S4 movement and activation of HCN and hERG results from channel-specific interactions between residues in the S4-S5 linker and the C-terminal end of the S6 domain. For hERG, we have characterized the gating currents associated with S4 movement and the structural basis for drug binding; we will extend these studies to HCN channels. In addition, the molecular mechanisms of altered hERG channel gating induced by changes in external cations will be determined. Ionic and gating currents will be recorded using two microelectrode and cut-open Vaseline gap voltage clamp techniques of channels heterologously expressed in Xenopus oocytes. These studies promise to further our understanding of the biological oscillator that controls heart rhythm.